Protein S is a key component for the regulation of hemostasis. Protein S serves as a cofactor for activated Protein C, an enzyme responsible for the degradation of two protein elements of the blood coagulation pathway. Consequently, Protein S through the action of Protein C down-regulates the blood clotting process. Individuals deficient in functional Protein S, either because of genetic or environmentally-acquired reasons, are at a higher risk of experiencing thrombotic disorders. Symptoms commonly associated with Protein S deficiency include thrombophlebitis, deep vein thrombosis, and pulmonary emboli. At the present time the genetic basis of Protein S deficiency and its relationship to thrombosis are unknown. The long-term objective of the proposed research is to understand the genetic basis of Protein S deficiency and thrombosis. This will be accomplished, in part, through the proposed studies, designed to provide a better understanding of the normal and abnormal Protein S genes and the functional properties of the Protein S products. Specific aims and methods for achieving these goals include: a) characterization of gaps in introns A, C and I of the expressed Protein S gene (PS-alpha), using PCR amplification and confirmation by genomic Southern hybridization; b.) cloning and characterization by DNA sequencing and Southern mapping of the PS-alpha gene from genetically Protein S deficient individuals; c) site-directed mutagenesis and mammalian cell expression studies to address structure/function relationships relating to the role(s) of N-glycosylation for Protein S. Comparative studies with deglycosylated plasma-derived Protein S will also be performed; and d) isolation and characterization with conventional biochemical methods of a plasma component that reduces the interaction between Protein S and C4b-binding protein. The results of these studies will lead to a better understanding of the structure and function of Protein S; and may eventually lead to improved methods of diagnosis and therapy for patients having Protein S deficiency and resulting thrombotic disorders.